The physiological stimulus for transmitter release is a depolarization-induced influx of calcium into the nerve terminal. The proposed experiments will use electrophysiological techniques to investigate fundamental questions concerning the mechanism of transmitter release from vertebrate motor nerve terminals. Using a new technique for recording intracellularly from intact, functional motor nerve terminals, we have observed prominent, slow, calcium-sensitive afterpotentials following action potentials in motor nerve terminals. We will determine the ionic mechanisms of these afterpotentials, and will characterize the kinetics and ionic specificity of the slow calcium conductance system (and of a possible calcium-sensitive, slow potassium conductance system) in tetrodotoxin-blocked motor nerve terminals. We will measure how several clinically important drugs affect this presynaptic calcium conductance. By recording presynaptic potentials simultaneously with end-plate potentials and miniature end-plate potentials, we will determine how activation of presynaptic conductance systems influences the magnitude and time course of transmitter release. We will increase the calcium permeability of nerve terminals by a variety of treatments and then measure the relationship between bath calcium concentration and transmitter release. We will try to determine whether the very nonlinear relationship between calcium concentration and transmitter release arises at calcium entry into the nerve terminal, at the reaction between calcium and release sites, or from a calcium-induced release of calcium from intraterminal membranes. We will also try to determine whether the calcium-dependent facilitation of transmitter release observed during repetitive stimulation is due to facilitated calcium influx into the nerve terminal, residual releasing factors, or internal calcium release. The ability to record presynaptic potential changes as well as the resulting transmitter release will allow us to test these various hypotheses far more directly than they could ever be tested before.